As in the past, most new FDA-approved drugs for cancer and other indications are derived from natural products or inspired by natural products. Accordingly, it is proposed here to screen the NCI collection of extracts to discover novel molecules that inhibit the deubiquitylating enzyme USP7 (HAUSP), a validated anticancer target from the ubiquitin proteasome pathway. USP7 works in cells to stabilize its various substrates, including the proto-oncogene HDM2 and the DNA repair protein claspin, permitting HDM2-enabled proteasomal degradation of the tumor suppressor p53 and activation of the G2M checkpoint, respectively. A selective inhibitor of USP7 is predicted to exert antitumor activity by stabilizing p53 and/or by ablating the G2M checkpoint in the presence of a genotoxic agent. Initial screening of the natural product extracts will be performed in cell based assays to eliminate the reactive tannins present in many of the extracts; HCT-116 cells stably transfected with a p21-luciferase construct reporting the transcriptional activation of p53 will be the first cll based screen. This screen has the advantage of identifying functionally relevant molecules. Hits from this screen will be counterscreened in a second cellular assay to monitor activity in a distinct USP7 regulated pathway to eliminate compounds that act on irrelevant pathways, and confirmed hits will be detanninized and evaluated for their ability to modulate the activity of purified USP7. Extracts with appropriate activity against USP7 will be queued for bioassay guided fractionation; active principles will be further evaluated by determination of their structures and their biochemical and cellular activities. In Phase II, selected inhibitors will undergo preclinical development as anticancer agents. PUBLIC HEALTH RELEVANCE: Both historically and at the present time natural products have been the major source of new drugs. Progenra will collaborate with the eminent natural products researcher Dr David Kingston of Virginia Tech to search for anticancer treatments from the ubiquitin-proteasome pathway, one of the most active new therapeutic target areas in molecular oncology. Inhibitors of the cancer-related enzyme USP7, a well-described component of this pathway, will be isolated from natural products extracts from the National Cancer Institute for development as novel therapies for neoplastic disease.